Long-term in vivo transgene expression

Abstract

Efficient and prolonged hCFTR expression is one of the major obstacles for cystic fibrosis lung therapy. hCFTR mRNA expression levels depend on eukaryotic expression cassette components, prokaryotic backbone elements, and the gene transfer method may also influence transcriptional silencing mechanisms. A codon-optimized and CpG-reduced human CFTR gene (CO-CFTR) was made. Various vector modifications were tested to facilitate extended duration of CO-CFTR expression. Insertion of an extended 3′BGH transcribed sequence (712 bp) in an inverted orientation produced prolonged expression of CO-CFTR expression at biologically relevant levels. Further studies revealed that prolonged CO-CFTR expression is dependant on the orientation of the extended BGH 3′ BGH transcribed sequence and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements.

1. A nucleic acid molecule comprising a portion, wherein said portion comprises at least 50 nt of SEQ ID NO: 1, wherein said portion is operably linked to a promoter from which transcription of the portion is initiated, wherein the portion is in inverted transcriptional orientation relative to its orientation in the bovine growth hormone (BGH) gene, wherein when the nucleic acid molecule further comprises a transgene operably linked to the promoter and located between the promoter and the portion, the portion improves long-term expression of the transgene. 
     
     
       2. The nucleic acid molecule of  claim 1  wherein the portion comprises untranslated sequences of the BGH gene. 
     
     
       3. The nucleic acid molecule of  claim 1  wherein the portion comprises translated sequences of the BGH gene. 
     
     
       4. The nucleic acid molecule of  claim 1  which spans the 3′ end of the open reading frame of the bovine growth hormone (BGH) gene and comprises both translated and untranslated sequences of the BGH gene. 
     
     
       5. The nucleic acid molecule of  claim 1  wherein the portion comprises the sequence shown in SEQ ID NO: 1. 
     
     
       6. The nucleic acid molecule of  claim 1  wherein the portion comprises the sequence shown in SEQ ID NO: 2. 
     
     
       7. The nucleic acid molecule of  claim 1  wherein the portion consists of the sequence shown in SEQ ID NO: 1. 
     
     
       8. The nucleic acid molecule of  claim 1  wherein the portion comprises the sequence shown in SEQ ID NO: 5. 
     
     
       9. The nucleic acid molecule of  claim 1  wherein the nucleic acid further comprises a transgene. 
     
     
       10. The nucleic acid molecule of  claim 9  wherein the transgene is shown in SEQ ID NO: 3. 
     
     
       11. The nucleic acid molecule of  claim 9  wherein the transgene is shown in SEQ ID NO: 4. 
     
     
       12. The nucleic acid molecule of  claim 1  wherein a restriction endonuclease site for inserting a transgene is 3′ of the promoter and 5′ of the portion. 
     
     
       13. The nucleic acid molecule of  claim 1  wherein the promoter is selected from the group consisting of human beta-actin, human polyubiquitin C, and SV40. 
     
     
       14. The nucleic acid molecule of  claim 1  which is an expression vector. 
     
     
       15. The nucleic acid molecule of  claim 1  which is a viral vector. 
     
     
       16. The nucleic acid molecule of  claim 1  wherein the portion is between 50 and 150 bases in length. 
     
     
       17. The nucleic acid molecule of  claim 1  wherein the portion is between 100 and 200 bases in length. 
     
     
       18. The nucleic acid molecule of  claim 1  wherein the portion is between 100 and 1000 bases in length. 
     
     
       19. The nucleic acid molecule of  claim 1  which is compacted to form a DNA nanoparticle. 
     
     
       20. A method of improving long-term expression of a transgene in a nucleic acid molecule, comprising:
 inserting into the nucleic acid molecule a portion comprising at least 50 nt of SEQ ID NO: 1, so that it is operably linked to a promoter from which transcription of the portion is initiated, wherein the portion is in inverted orientation relative to its orientation in the BGH gene and wherein the transgene is operably linked to the promoter between the promoter and the portion. 
 
     
     
       21. The nucleic acid molecule of  claim 1  wherein the portion consists of the sequence shown in SEQ ID NO: 2. 
     
     
       22. The nucleic acid molecule of  claim 1  wherein the portion consists of the sequence shown in SEQ ID NO: 5.